用于检测和定量食品中金黄色葡萄球菌肠毒素B的免疫定量实时聚合酶链反应
Immunoquantitative real-time PCR for detection and quantification of Staphylococcus aureus enterotoxin B in foods.
作者信息
Rajkovic Andreja, El-Moualij Benaissa, Uyttendaele Mieke, Brolet Philippe, Zorzi Willy, Heinen Ernst, Foubert Ellen, Debevere Johan
机构信息
Laboratory of Food Microbiology and Food Preservation, Department of Food Safety and Food Quality, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium.
出版信息
Appl Environ Microbiol. 2006 Oct;72(10):6593-9. doi: 10.1128/AEM.03068-05.
A real-time immunoquantitative PCR (iqPCR) method for detection of Staphylococcus aureus enterotoxin B (SEB) was developed and evaluated using both pure cultures and foods. The assay consisted of immunocapture of SEB and real-time PCR amplification of the DNA probe linked to the detection antibody. iqPCR was compared to an in-house enzyme-linked immunosorbent assay (ELISA) using the same couple of capture-detection antibodies and to commercial kits for detection of S. aureus enterotoxins (SE). The iqPCR was approximately 1,000 times more sensitive (<10 pg ml(-1)) than the in-house ELISA and had a dynamic range of approximately 10 pg ml(-1) to approximately 30,000 pg ml(-1). iqPCR was not inhibited by any of the foods tested and was able to detect SEB present in these foods. No cross-reactivity with SE other than SEB was observed. Application of iqPCR for detection of SEB in cultures of S. aureus revealed the onset of SEB production after 4 h of incubation at 22, 37, and 42 degrees C, which was in the first half of the exponential growth phase. The total amounts of SEB produced by the two strains tested were larger at 42 degrees C than at 37 degrees C and were strain dependent.
开发了一种用于检测金黄色葡萄球菌肠毒素B（SEB）的实时免疫定量PCR（iqPCR）方法，并使用纯培养物和食品进行了评估。该检测方法包括对SEB进行免疫捕获以及对与检测抗体相连的DNA探针进行实时PCR扩增。使用相同的一对捕获-检测抗体，将iqPCR与内部酶联免疫吸附测定（ELISA）以及用于检测金黄色葡萄球菌肠毒素（SE）的商业试剂盒进行了比较。iqPCR的灵敏度比内部ELISA高约1000倍（<10 pg ml⁻¹），动态范围约为10 pg ml⁻¹至约30,000 pg ml⁻¹。iqPCR不受任何测试食品的抑制，并且能够检测出这些食品中存在的SEB。未观察到与SEB以外的其他SE有交叉反应。应用iqPCR检测金黄色葡萄球菌培养物中的SEB发现，在22、37和42℃孵育4小时后开始产生SEB，这处于指数生长期的前半段。所测试的两种菌株产生的SEB总量在42℃时比在37℃时更大，并且具有菌株依赖性。
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